A one-step molecular biology method for simple and rapid detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 strain.

Six reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primers designed against conserved regions of segment 6 (s6) gene were used for the detection of grass carp Ctenopharyngodon idella reovirus (GCRV) HZ08 subtype. The entire amplification could be completed within 40 min at 62·3° C. The RT-LAMP showed higher sensitivity than reverse-transcription polymerase chain reaction (RT-PCR). The RNA detection limit was 10 copies µl⁻¹ for RT-LAMP assay and 100 copies µl⁻¹ for conventional RT-PCR. In specificity tests, no cross-reactivity was detected in other viruses from common aquatic animals. In addition, the reaction results can be visualized by using calcein fluorescent dye. Furthermore, a total of 86 samples were tested by RT-LAMP, RT-PCR and virus isolation. The results demonstrated that all 54 specimens identified as positive by virus isolation were also positive when detected by RT-LAMP. Seven out of 54 samples, however, were misidentified by RT-PCR. The RT-LAMP method is more accurate than conventional RT-PCR. The results indicate that RT-LAMP has potential as a simple and rapid diagnosis technique for the detection of GCRV HZ08 subtype infection.

Effects of six APOA5 variants, identified in patients with severe hypertriglyceridemia, on in vitro lipoprotein lipase activity and receptor binding.

OBJECTIVE: The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible. METHODS AND RESULTS: We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant. CONCLUSIONS: The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression.

Significance of IL28RA in diagnosis of early pancreatic cancer and its regulation to pancreatic cancer cells by JAK/STAT signaling pathway - effects of IL28RA on pancreatic cancer.

OBJECTIVE: To explore the significance of IL28RA in diagnosis of early pancreatic cancer and its regulation to pancreatic cancer cells by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. PATIENTS AND METHODS: A total of 81 patients with early pancreatic cancer were enrolled as a pancreatic cancer group, and 81 patients with benign pancreatic diseases were enrolled as a benign disease group. Western blot was adopted to analyze the serum IL28RA expression of the two groups and its diagnostic value in early pancreatic cancer. A pancreatic cancer cell model was constructed, and the IL28RA expression in pancreatic cancer cells, PANC-1 and BXPC-3, was up-regulated to explore the biological function of pancreatic cancer cells after up-regulation of IL28RA and the effects on JAK-STAT signaling pathway. RESULTS: Lowly expressed in serum of patients with pancreatic cancer, IL28RA showed a sensitivity of 80.25%, specificity of 75.31%, and area under the curve (AUC) of 0.846 in diagnosis of early pancreatic cancer. It was found that up-regulation of IL28RA expression in pancreatic cancer cells inhibited proliferation and invasion abilities of pancreatic cancer cells, increased apoptosis rate and expression of pro-apoptotic protein bax, decreased expression of anti-apoptosis protein bcl-2, and significantly inhibited phosphorylation level of JAK2 and STAT3 proteins. CONCLUSIONS: IL28RA is lowly expressed in pancreatic cancer patients, and has certain diagnostic value for early pancreatic cancer. Its up-regulated expression can inhibit the proliferation and invasion of pancreatic cancer cells, and promote their apoptosis by inhibiting the activation of JAK-STAT signaling pathway.

Neural stem/progenitor cells survive and differentiate better in PD rats than in normal rats.

To investigate the effects of grafted neural stem/mesencephalic progenitor cells (NSCs/MP) on rotational behavior of Parkinson's disease (PD) rats and the influence of intracerebral environment on NSCs/MP, we observed the survival and differentiation of NSCs/MP transplanted into 6-hydroxydopamine (6-OHDA)-lesioned and intact striatums. NSCs/MP were prepared from E(11-15) rats and proliferated in serum-free medium with bFGF for several weeks. One day after being primed with serum/dbcAMP to differentiate, cell suspensions were grafted into 6-OHDA-lesioned and intact striatums respectively. It had been found that NSCs/MP were able to survive better and differentiate into more tyrosine hydroxylase (TH)-positive neurons in 6-OHDA-lesioned striatums than in intact ones, and apomorphine-induced rotations were obviously attenuated in MP graft models. The data suggested that NSCs/MP tend to survive and differentiate into TH-positive neurons in 6-OHDA-lesioned striatums. The data demonstrated that striatums in which DAergic terminals are destroyed by 6-OHDA undergo some changes and thus provide more appropriate conditions for NSCs/MP to differentiate into mature DAergic neurons. Furthermore, the finding that MP had greater relieving effects on rotational behavior than NSCs suggests that NSCs could not be used in clinical therapy of PD unless being induced into MP in vitro before transplantation.

Mesencephalic progenitors can improve rotational behavior and reconstruct nigrostriatal pathway in PD rats.

The aim of this study was to investigate the possibility of mesencephalic progenitors (MP) in treating Parkinson's disease (PD). MP were prepared from E(11-13) rats and proliferated in serum-free medium with basic fibroblast growth factor (bFGF) for 10 days. Cells were then collected and implanted into the striatum only--single grafts or simultaneously into the substantia nigra (SN) and the striatum--double grafts. Twelve weeks after transplantation, DiI, a fluorescent dye, was microinjected into the ipsilateral striatum. Using this strategy, it was found that MP of double grafts had more potent effects on rotational behavior than that of single grafts. Injection of the retrograde tracer DiI into the striatum resulted in fluorescent-labeled cells within the intranigral grafts in double grafts. These data greatly support that MP transplants could not only improve rotational behavior, but does help to re-establish nigrostriatal connections so that it may become one efficient way in treating PD.